Multiple signals determine cell fates such as cell birth, death, and differentiation during development and in adult multicellular organisms. A major challenge in biology is to understand how signals from different receptors are integrated to determine an appropriate response. This process is particularly complicated in migrating cells such neurons and neural crest cells, and may go awry, resulting in increased cell proliferation or migration in cancer. Neuroblastoma cell lines provide a model system to study the molecular mechanisms involved in sorting and transactivation between receptors. We hypothesize that receptor sorting into endosomes has functional significance for the intracellular location and duration of signaling and that signaling from endosomes plays a major role in compartmentalizing or integrating signals from different receptors. Specifically, we hypothesize that multiprotein complexes of receptors and their effectors in endosomes play a role in receptor crosstalk, and that receptor crosstalk or transactivation may be facilitated when two receptors are in the same endosome. We have recently shown that three different types of receptors are localized predominately in endosomes that are resolved from one another using a high-resolution organelle fractionation method based on mass and density. The data suggest that receptor sorting into specific signaling endosomes affects the compartmentalization of signaling pathways. We propose experiments to ask three questions about receptor sorting and transactivation in endosomes. First, how are two receptors sorted from one another into specific signaling endosomes? The two receptors for nerve growth factor (NGF), TrkA and p75NTR, are rapidly sorted upon ligand binding to distinct endosomes. Based on preliminary data, we hypothesize that sorting of these two receptors away from one another involves dynamic interactions between detergent-insoluble lipid rafts and microtubules. We propose to analyze interactions between TrkA, p75NTR and microtubules using fractionation of organelles and molecular complexes in lipid rafts, and in live cells using fluorescent protein constructs and microscopy. Second, is there crosstalk or transactivation between TrkA and another receptor tyrosine kinase, anaplastic lymphoma kinase (ALK)? ALK is frequently mutated in neuroblastoma, highly tyrosine phosphorylated in neuroblastoma cell lines, and appears to be activated in neuroblastoma endosomes, indicating an important role in the choice between proliferation and differentiation. We will investigate whether the phosphorylation of TrkA is affected by ALK and vice versa, and whether this translates into a functional response from one or the other receptors. Third, we will ask if ALK and TrkA are in the same or different endosomes in neuroblastoma cells using fluorescence microscopy and cell fractionation followed by immunoisolation of specific endosomes. We will also determine the components of ALK and TrkA endosomes. Together, these experiments explore molecular mechanisms that are relevant to our understanding of basic developmental biology and cancer. PUBLIC HEALTH RELEVANCE: Neuroblastoma is at present largely refractory to therapy. Thus, the clear challenge is to devise an effective therapy for this intractable cancer, which would have a worldwide impact on child health. A detailed understanding of the molecular mechanisms that underlie key differentiation steps in neural crest and neuroblastoma will potentially identify pharmacologically accessible factors or pathways for new therapeutic approaches. These studies are also relevant to the use of stem cells for nerve regeneration.